This invention relates generally to the measurement of light created by the absorption of high intensity light of one wavelength range and subsequent re-emission at another wavelength range, and specifically to an apparatus designed to conduct bioanalytical assays by measurement of the emitted light.
Bioanalytical binding assays are widely employed by the pharmaceutical, drug-discovery and biotechnology industries. A variety of such binding assays have been developed, including but not limited to, receptor binding, enzyme activity and inhibition, protein-protein interactions, and nucleic acid hybridization. The specific techniques used to perform these assays can be generally classified as heterogeneous or homogenous.
Heterogeneous assays involve the physical separation of reacted and unreacted species prior to sample analysis. The separation is usually effected by preferential adsorption of assay components onto a suitable solid support, followed by washing of that support. A related technique involves filtration of the assay mixture through an appropriate trapping medium. These separation techniques typically result in decreased assay accuracy, decreased assay precision and an increase in labor requirements, all due to the addition of a separation step.
Homogeneous assays involve the analysis of assay mixtures without the physical separation of reacted and unreacted species. Since homogeneous assays have fewer steps, the assay accuracy and precision are higher than their heterogeneous counterparts. Moreover, labor costs are reduced. However, homogeneous assays suffer from inherent interference problems. The interference occurs as a result of the simultaneous measurement of both reacted and unreacted species. Other potential interference problems include those caused by biological matrix effects and extraneous light production from system components. These interference problems may significantly reduce the usable dynamic range, accuracy, and precision of any assay.
Both heterogeneous and homogeneous assays can use a variety of labeling and detection technologies, including but not limited to, radioisotopic, fluorometric, luminometric, and colorimetric methods. The use of these technologies is well documented in the technical literature and well known to those skilled in the art. These technologies suffer from certain limitations and disadvantages, including poor sensitivity, poor dynamic range, occupational safety risks, signal instability, and high cost.
U.S. Pat. No. 5,340,716 (Ullman, et. al. xe2x80x9cAssay Method Utilizing Photoactivated Chemiluminescent Labelxe2x80x9d) describes an alternative assay technology that possesses many of the advantages of a homogeneous system, while eliminating many of the interferences associated with traditional photon measurement technologies. The Ullman et al. patent describes the use of Luminescence Oxygen Channeling Immunoassay (xe2x80x9cLOCIxe2x80x9d) procedures for conducting a variety of clinically important assays. Luminescent Oxygen Channeling Immunoassays can be used for quantitative determination of a variety of analytes in a wide range of concentrations. Such analytes include unbound drugs, DNA and immunoglobulins. The methods for carrying out these immunoassays have also been described in various technical publications. See, e.g., Edwin F. Ullman et al., xe2x80x9cLuminescent Oxygen Channeling Immunoassay (LOCI(trademark)): sensitive, broadly applicable homogeneous immunoassay methodxe2x80x9d; Clinical Chemistry 42:9, pp. 1518-1526 (1996); Edwin Ullman, xe2x80x9cLuminescent Oxygen Channeling Immunoassay (LOCI) for Human Thyroid Stimulating Hormonexe2x80x9d, in xe2x80x9cBioluminescence and Chemiluminescence, Proceedings of the 8th International Symposium on Bioluminescence and Chemiluminescencexe2x80x9d, Cambridge, September, 1994 (John Wiley and Sons). In the clinical laboratory, these assays are typically conducted in test tubes at sample volumes approaching 1 mL.
The pharmaceutical, drug-discovery and biotechnology industries, along with bioanalytical instrumentation vendors, have established the microplate as the preferred format in which to perform assays. This device is a plastic plate containing multiple wells in a regular array, each of which may contain a unique sample. The current standard is the 96-well plate, and assays are typically conducted in an approximate volume of 200 uL per well. As both reagent costs and sample throughput requirements increase, there is a need for an assay technology and measurement apparatus which have good precision and accuracy, high sensitivity, and can be converted to lower volume formats such as those used in 384-well and 1536-well microplates. There is also an existing need for integrated assay reaction and reading analyzers capable of operation in the nanoliter to microliter volume range.
Thus, one object of the present invention is to provide a compact apparatus that can be used in conjunction with an accurate method for producing and detecting light in liquid samples in microplate wells.
Another object of the present invention is to provide an apparatus for conducting extremely sensitive assays in very small sample volumes in high well density microplate formats.
Another object of the present invention is to provide a compact apparatus for producing and detecting light generated by biological samples using the LOCI technique, wherein the amount of light is proportional to the quantity of analyte present in the sample.
A further object of the present invention is to provide a compact apparatus for conducting bioanalytical assays using the LOCI technique by excitation of the liquid sample contained in an opaque well.
Another object of the present invention is to provide an apparatus for conducting bioanalytical assays using the LOCI technique for samples in the microliter to nanoliter volume range.
Yet another object of the present invention is to provide an apparatus for conducting bioanalytical assays using the LOCI technique in an automated fashion using microplates with 96, 384, or 1536 wells, or a labchip consisting of multiple channels.
A further object of the present invention is to provide an apparatus for conducting the aforementioned assays at a high speed by simultaneously measuring multiple wells on the microplate or channels on the labchip.
In accordance with one aspect of the present invention, a system for measuring light emitted by a sample in a microplate well, is presented. This invention includes a light source, which produces light at a first wavelength band (the excitation wavelength band), a bifurcated fiber bundle which transmits the excitation light to the sample and the emitted light to the detector, a lens which directs the excitation light into the sample and collects the light emanating from the sample at a second wavelength band (the emission wavelength band), optionally, an emission band-pass filter which blocks extraneous light, including that which corresponds to the excitation wavelength band, while passing light in the emission wavelength band, a shutter which protects the detector, thus allowing the introduction of more excitation light into the sample, and a light detector.
Unlike traditional systems of this general type, the excitation wavelength band is at a longer wavelength, and the emission wavelength band is at a shorter wavelength. Moreover, the light emanating from the sample is collected after the excitation light source is turned off. These characteristics result in lower non-specific light production, thus enhancing the sensitivity of the system.
As a result of the use of fiber bundles which transmit light to and from the sample, the light source and detector may be placed remotely to the sample. This further permits the use of multiple sets of light sources and detectors, which provides a means for simultaneously quantifying samples in different wells, thus increasing throughput.
In accordance with another aspect of the present invention, a method for measuring light emanating from a sample, produced by excitation from a high intensity light source, is presented. This invention utilizes a system for the production of high intensity light at the excitation wavelength band, which passes the high intensity light through a fiber bundle into a sample in a microplate well. The light is emitted by the liquid subjected to the excitation light as the result of the presence of materials, such as a chemiluminescent material, through the fiber bundle and is directed through an emission band-pass filter that eliminates any extraneous light, including that which corresponds to the excitation wavelength band. The light is then passed through a shutter and to a light detector, such as a photomultiplier tube. A controller intermittently activates a light source to produce high intensity light and closes a shutter whenever the high intensity light is passed through the system. This controller could be a mechanical or electronic shutter or a light source gating circuit.
In accordance with a further aspect of the present invention, an analyzer for carrying out Luminescence Oxygen Channeling Immunoassays in microplates, is presented. A biological binding reaction is carried out in liquid samples in microplate wells. A high intensity light is introduced into the well. The light initiates a photochemical reaction that results in production of light of a different wavelength. The intensity of this light is proportional to the amount of the analyte present in the sample. The emitted light resulting from the photochemical reaction has a wavelength band which is shorter than the excitation wavelength band. The emitted light is collected by a lens and a fiber bundle located above the sample, and is then passed through a filter, which eliminates extraneous light, including that which corresponds to the excitation wavelength band. The light transmitted through the filter passes through an aperture in the shutter and is measured by a detector, such as a photomultiplier tube. A controller activates the light source and closes the shutter whenever the light source is activated.